Modification of nuclear and cytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is as abundant and as dynamic as protein phosphorylation in all metazoans. The dynamic interplay between O-GlcNAc and O-phosphate plays a key role cellular signaling, transcription and in the functions of nuclear oncogene and tumor suppressor proteins. O-GlcNAc also plays a key role in protecting cells from varied forms of stress, suggesting that the saccharide is important to tumor cell survival mechanisms. Our long-term goal is to elucidate the functions and interplay between O-GlcNAc and O-phosphate important to the oncogenic and metastatic phenotype. This application builds upon years of methods development to directly determine the molecular roles of O-GlcNAc on c-Myc oncogene protein, Rb tumor suppressors and in the cellular mechanisms protecting cells from extreme stresses. Specific Aims are: Aim 1: Elucidate the functions of O-GIcNAc On the c-Myc oneogene protein. A. Quantify the dynamic modification of c-Myc's transactivation domain (TAD) as a function of growth, cellcycle, apoptosis, & stress using CE-laser flourecence-MS/MS. B. Determine the functions of c-Myc TAD isoforms using mutant c-Myc and intein-produced isoforms. C. In vitro physical analyses of the structures of c-Myc TAD isoforms. D. Comparative kinetic analyses of O-GlcNAc Transferase, GSK3( and MAP kinases on glyco/phospho forms of the c-Myc TAD region. Aim 2: Determine the functions of O-GIcNAc on the tumor suppressor Rb and Rb p107 proteins. A. Map O-GlcNAc Sites. B. Quantify changes in OGlcNAc/O-phosphate during cell-cycle, cell growth and apoptosis. C. Comparative analyses of Rb & p107 isoforms protein:protein interactions and transcriptional activities. Aim 3: Determine the roles of O-GIcNAc in protecting cells from stress. A. Mechanisms upregulating O-GlcNAc in response to stress. B. Functions of O-GlcNAc on HSP70. C. Elevated O-GlcNAc affects on apoptosis and protein aggregation? These studies are elucidating molecular mechanisms of oncogenesis and metastasis, over-looked by others, that could lead to unexpected therapeutic approaches.